Enteroviruses cause a diverse spectrum of human diseases including conjunctivitis, myocarditis, aseptic meningitis, acute flaccid paralysis, and fatal systemic infections of neonates. Poliovirus, the prototypic enterovirus, is well characterized at the molecular level and still serves as the most appropriate virus for detailed studies of RNA translation and RNA replication. In this proposal, poliovirus mRNA translation and RNA replication will be studied in cell-free reactions capable of supporting sequential translation and replication of poliovirus RNA. These reactions are advantageous because they support authentic translation and replication of poliovirus RNA while providing numerous technical advantages including the ability to synchronize viral mRNA translation and viral RNA replication. Using these reactions, we discovered that a cis-active RNA structure at the 5' terminus of poliovirus RNA interacts with seemingly distal cis-active structures in the viral open reading frame and the 3' nontranslated region to coordinately regulate the efficient translation and replication of poliovirus RNA. Partially characterized temporally dynamic ribonucleoproteins containing these cis-active RNA structures mediate and regulate the sequential steps of poliovirus mRNA translation and RNA replication. The experiments described in this proposal will characterize these ribonucleoproten complexes more thoroughly. Experiments will be performed to: 1) determine how the 5' cloverleaf RNA structure of poliovirus potentiates viral mRNA translation, 2) determine how translating ribosomes regulate, in part, the switch from viral mRNA translation to RNA replication, 3) identify the seemingly distal cis-active RNA structures that interact to regulate sequential steps of viral RNA replication, and 4) determine how protein 2C ATPase mediates interactions between seemingly distal cis-active ribonucleoprotein complexes. These studies will contribute substantial new information to support the popular new paradigm of 5'-3' RNA interactions in messenger ribonucleoprotein complexes and RNA replication complexes. The experiments directly test a hypothesis concerning the mechanism by which RNA replication machinery avoids ribosome-replicase collisions. The experiments also test a new, theoretically appealing hypothesis that clearly explains the mechanisms controlling asymmetric RNA replication. [unreadable] [unreadable]